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First published online February 1, 2008
Journal of Experimental Biology 211, 599-605 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.009225
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Reconstitution of a chemical defense signaling pathway in a heterologous system

Staci A. Padove Cohen1, Hanns Hatt2, Julia Kubanek3 and Nael A. McCarty1,*

1 Biology, Georgia Institute of Technology, Atlanta, GA 30332, USA
2 Cell Physiology, Ruhr-Universitaet Bochum, Bochum 44780, Germany
3 Chemistry and Biochemistry, Biology, Georgia Institute of Technology, Atlanta, GA 30332, USA


Figure 1
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  		Fig. 1. G{alpha}s signaling pathway utilized in bioassay. The cyclic AMP-dependent response in oocytes co-expressing the zebrafish cDNA library and CFTR is activated by the binding of a ligand to a membrane receptor, causing the receptor to interact with a G protein (G{alpha}s). Upon dissociation from the heterotrimeric G-protein–receptor complex, the {alpha} subunit activates adenylyl cyclase. This action leads to a cAMP signaling cascade, ending in the activation of CFTR. The output of this cascade is measured by electrophysiology and is the basis of the functional assay.

 

Figure 2
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  		Fig. 2. Receptor-mediated responses in oocytes expressing zebrafish cDNA library or OR-I7. (A) Current changed in response to IBMX, an activator of CFTR, but did not change in response to octanal, an agonist of the OR-I7 chemoreceptor, in cells heterologously expressing CFTR alone. (B) Both forskolin, an activator of CFTR, and octanal caused increased CFTR activity when applied to oocytes expressing OR-I7 and CFTR. (C) Both isoproterenol (Iso), an agonist of β2AR, and octanal caused increased CFTR activity when applied to oocytes expressing the library and CFTR but caused no change in oocytes expressing CFTR alone (data not shown). (D) Oocytes expressing only the library had a very slight change in current in response to formoside. Note the difference in time scale from A to D.

 

Figure 3
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  		Fig. 3. Electrophysiological responses to chemical deterrents. No electrophysiological change was seen in response to application of oroidin (A) or sceptrin (B). A mixture of ectyoplasides A and B caused a slight change in current (C), indicating activation of CFTR via the Gs signaling pathway, which was not observed in control cells (D). Iso, isoproterenol; Oct, octanal.

 

Figure 4
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  		Fig. 4. Formoside induces an electrophysiological response in zebrafish cDNA library-expressing oocytes. Formoside (Form) caused a response in library-expressing cells after activation of the Gs pathway with isoproterenol (Iso; A). No responses to formoside or octanal (Oct) were seen in oocytes without library (B). Note the difference in time scale between A and B. Inset shows the response to isoproterenol in B on the same time scale as A.

 




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